高效的雪旺细胞体外培养方法

目的 介绍有效地获得大量高纯度、高活性的雪旺细胞培养纯化方法。方法 选用2～3天的SD大鼠双侧臂丛神经和坐骨神经，在解剖镜下剥离除去神经外膜，剪碎后采用高浓度酶消化法种植培养。随后阿糖胞苷连续作用3天去除成纤维细胞，同时逐渐降低培液中胎牛血清浓度。并使用超低酶浓度消化传代。结果本方法可从新生鼠获取近4×10^6雪旺细胞，成活率为98％；S-100免疫荧光法鉴定雪旺细胞纯度达96％以上。传统方法获得约3×10^6雪旺细胞，成活率为92％；雪旺细胞纯度为90％。结论 本方法可以获得大量纯度高、活性好的雪旺细胞，值得进一步推广。     

改良雪旺细胞传代培养的实验研究

目的:采用改良传代法进行雪旺细胞培养,以获取大量纯净的雪旺细胞用于周围神经组织工程研究。方法:采用4-5 d SD乳鼠坐骨神经和臂丛神经,运用双酶消化法结合单酶消化法进行雪旺细胞的原代培养。5-7 d后,采用单酶快速消化离心法进行传代培养,同时纯化雪旺细胞。结果:SABC免疫组化染色鉴定和计数板计数,改良传代法使体外培养的雪旺细胞纯度达98％左有,两种方法之间有显著性差异,P<0.001。MTT检测雪旺细胞生存和增殖时间延长1-2周左右。结论:与传统的传代培养方法相比,改良法既省时又避免了外源性物质如Ara-C对雪旺细胞的毒性作用,同时有利于雪旺细胞活力和纯度的提高。     

一种简便的雪旺细胞培养方法

目的介绍一种简便的雪旺细胞培养方法。方法采用反复组织块种植法,进一步在2.5%胎牛血清条件下抑制成纤维细胞生长,进行雪旺细胞培养;以S-100抗体染色鉴定雪旺细胞。结果获得纯度在98%以上的大量高活性雪旺细胞。结论本方法可以简便的获得较高纯度的雪旺细胞,值得进一步推广。     

利用贴壁特点提取和纯化乳鼠雪旺细胞

目的主要利用乳鼠的雪旺细胞比成纤维细胞贴壁快的特点,介绍简单而快速提取和纯化乳鼠雪旺细胞的方法。方法取3天SD大鼠双侧坐骨神经,在解剖镜下剥离去除神经外膜,剪碎后用Ⅰ型胶原酶消化,差速法去除成纤维细胞,分小皿培养进一步纯化细胞;光镜下计算细胞纯度;S-100免疫荧光鉴定细胞性质。结果获取的雪旺细胞纯度大于96%;S-100免疫荧光鉴定细胞为阳性。结论本方法可以简单而快速地获得高纯度雪旺细胞。     

一种新的组织块培养新生大鼠雪旺细胞方法(英文)

目的:为了建立一种组织块培养雪旺细胞的有效方法,为周围神经组织工程修复的研究提供种子细胞。方法:本研究从新生大鼠的周围神经分离雪旺细胞,经组织块法培养并纯化,观察并记录其形态特征;经HE染色以及S-100免疫组化染色鉴定并计算其纯度。结果:结果显示,4 d后,获得的雪旺细胞突起呈双极或三极,S-100阳性细胞纯度达到95%以上。结论:上述结果表明该方法快速有效,所获得的雪旺细胞纯度高、活性好。     

雪旺细胞体外培养的研究进展

近年来,人们研究发现,雪旺细胞(SC),作为周围神经系统的主要胶质细胞,其功能极其活跃,它能分泌多种神经营养因子及轴突引导因子,维持周围微环境的稳定,防止神经元胞体死亡和促进神经元轴突的再生;合成和分泌诸如层黏连蛋白(LN),纤维黏连蛋白(FN)及Ⅰ、Ⅲ和Ⅴ型胶原等细胞外基质和细胞黏附因子,与神经系统的发育和再生有着密切的关系,尤其是在神经系统损伤后功能恢复上有着重要的作用。     

人体正中神经源雪旺细胞培养技术

雪旺细胞 (ＳＣ)是周围神经系统中一种功能特殊的胶质细胞 ,它不仅与周围神经发生、发育以及形态、功能密切相关 ,而且在周围神经受损后修复与再生过程中起重要作用。研究表明ＳＣ一方面能产生多种神经营养因子 ,这些神经营养因子在神经受损后能维持神经元存活 ;另一方面ＳＣ可以产生细胞外基质、细胞粘着分子 ,对神经轴突再生起支持和引导作用 ,为神经轴突提供适宜再生的微环境[1] 。ＳＣ的培养是研究ＳＣ及其产物的作用的前提 ,也为临床用于周围神经受损后修复、再生提供ＳＣ移植的材料来源[2 ] 。然至今动物实验很多[4] ,而人体ＳＣ培养…     

高糖对体外培养雪旺细胞影响的研究进展

雪旺细胞在糖尿病周围神经病变过程中起着重要的作用，近年来伴随组织细胞体外培养技术的日趋成熟，探讨体外高糖环境对雪旺细胞的影响逐渐成为研究新热点。本文就近年来高糖环境对体外培养雪旺细胞影响的相关研究作一综述。     

激活态雪旺细胞在胶元几丁糖膜上生长规律的实验研究

目的 探索成年SD大鼠激活态雪旺细胞(SC)在体外在胶元几丁糖膜上的生长规律。方法 成年SD大鼠的坐骨神经切断预变性7天,采用复合酶分步消化法,接种于胶元几丁糖膜上,通过相差显微镜和扫描电镜观察细胞生长情况,S-100染色鉴定细胞的纯化程度。结果 200μl浓度为2×10~4/ml的激活态的SCs接种于胶元几丁糖膜上和培养皿上,2周后细胞浓度分别达到30×10~4/ml和20×10~4/ml,相差显微镜和扫描电镜下观察显示SCs在胶元几丁糖膜上贴壁生长情况良好,体外倍增时间为4天。绘制其生长曲线。结论 胶元几丁糖膜和高度纯化的激活态雪旺细胞有良好的亲和性,以其为工艺材料制成的组织工程支架很有可能在促进周围神经再生中发挥优势作用。     

格林巴利血清蛋白对体外培养的雪旺细胞的影响

目的：探讨格林巴利血清蛋白对聚酯纤维上雪旺细胞粘附力的影响。材料和方法：实验组和对照组分别用含格林巴利血清蛋白的培养液和含健康者血清蛋白的培养液培养聚酯纤维网架上SD大鼠的雪旺细胞（SchwannCellSC);常规培养组用小牛血清培养液培养,在培养60min,120min,4h,8h,24h,72h观察SC的形态和对纤维粘附力的变化。结果：和常规培养组比较,对照组SC无明显差异;实验组从120min到8h,已包卷纤维的单个SC或SC链由梭形变为球形或者聚集成团;最后,绝大部分SC溶解成碎屑漂浮在纤维旁;24h,有少量新的SC从神经段迁移到纤维上。结论：格林巴利血清蛋白降低SC的粘附力是髓鞘脱失的重要因素。     

乳鼠雪旺氏细胞的纯化培养研究

目的：获得高纯度的乳鼠雪旺氏细胞。方法：采用出生后３～５天乳鼠的坐骨、臂丛神经，植块法培养乳鼠雪旺氏细胞；并通过精细剥除神经外膜、组织块反复再植、低浓度胰酶快速消化、差速贴壁法的有机结合对雪旺氏细胞进行纯化；继用抗Ｓ－１００蛋白单抗通过间接免疫荧光法及ＡＢＣ法进行细胞鉴定。结果：首次植块获得的细胞经纯化后雪旺氏细胞可高达８５％以上，反复植块者可达９５％，甚至高达９８％。结论：本方法简单、稳定，所获得的雪旺氏细胞纯度高、数量大、受非实验因素影响小     

模拟微重力对许旺细胞三维培养影响的初步研究

目的 初步探讨模拟微重力对许旺细胞三维培养的影响。方法 分别把粘附在聚羟基乙酸支架上生长３、７、１０及１４天的许旺细胞和新鲜分离的原代许旺细胞悬液连同聚羟基乙酸支架送入转壁式生物反应器（ＲＷＶＢ）中旋转培养５天,观察细胞在支架上的生长情况及超微结构的改变。结果 粘附在支架上的许旺细胞进入模拟微重力环境后细胞变圆变小,核染色质聚集,细胞器水肿护张,核／浆比例明显增大,微畿微管公散紊乱,细胞在３天内快速凋亡或碎裂溶解;尚示贴壁的原代许旺细胞进入模拟微重力环境后在支架表面均匀贴壁生长、增殖,蛋白合成活跃,个别细胞内可见髓鞘样结构,５天内未见细胞死亡现象。结论 贴壁生长３～１４天的许旺细胞进入模拟微重力环境后表现为快速凋亡、溶解,而未贴壁的原代许旺细胞进入模拟微重力环境后则功能活跃表达及快速增殖,可用于构建比较理想的许     

Models and methods to study Schwann cells

Schwann cells (SCs) are fundamental components of the peripheral nervous system (PNS) of all vertebrates and play essential roles in development, maintenance, function, and regeneration of peripheral nerves. There are distinct populations of SCs including: (1) myelinating SCs that ensheath axons by a specialized plasma membrane, called myelin, which enhances the conduction of electric impulses; (2) non‐myelinating SCs, including Remak SCs, which wrap bundles of multiple axons of small caliber, and perysinaptic SCs (PSCs), associated with motor axon terminals at the neuromuscular junction (NMJ). All types of SCs contribute to PNS regeneration through striking morphological and functional changes in response to nerve injury, are affected in peripheral neuropathies and show abnormalities and a diminished plasticity during aging. Therefore, methodological approaches to study and manipulate SCs in physiological and pathophysiological conditions are crucial to expand the present knowledge on SC biology and to devise new therapeutic strategies to counteract neurodegenerative conditions and age‐derived denervation. We present here an updated overview of traditional and emerging methodologies for the study of SCs for scientists approaching this research field.     

间充质干细胞体外分化为类许旺细胞的研究进展

目的：探讨化学合成物联合添加细胞因子的诱导方法诱导间充质干细胞体外分化为类许旺细胞的研究进展。方法：通过查阅国内外相关文献,从添加的化学合成物、各种细胞因子及诱导方案三方面来探讨间充质干细胞体外分化为类许旺细胞的相关研究,并进行分析总结。结果：化学合成物联合添加细胞因子诱导的三种方案都可以诱导间充质干细胞在体外分化具有与许旺细胞相似生物学特性及生理功能的类许旺细胞,其中以传统方法使用较多。结论：通过对诱导剂及诱导方案的总结与分析,初步探讨了间充质于细胞在体外定向诱导分化成为类许旺细胞的过程和影响因素,为进一步探讨间充质干细胞分化调控机制、分化能力及周围神经缺损的替代修复奠定实验基础。     

Purification and transfection of cochlear Schwann cells

Schwann cells line nerve fibers in the peripheral nervous system (PNS) and synthesize myelin. In addition, they support neuronal survival, neurite growth and regeneration. In dissociated cultures of postnatal mouse spiral ganglia, regenerating neurites spontaneously associate with Schwann cells. However, the mechanisms and consequences of interactions between cochlear Schwann cells and spiral ganglion neurites have not been examined. Further, the similarities and differences between cochlear Schwann cells and other PNS Schwann cells have not been studied. Experiments to examine these questions will rely on the ability to purify and characterize cochlear Schwann cells. Here we present methods for purifying Schwann cells from postnatal mouse cochleas and for transfecting them with expression plasmids. Dissociated spiral ganglia were plated on poly-d-lysine/laminin in medium containing neurotrophins, leukemia inhibitory factor (LIF), N2 supplement and serum and maintained for 5 days. Cells were harvested with trypsin/EDTA and subjected to an immuno-magnetic purification procedure. After 24 h in vitro, cultures were >85% Schwann cells. Nucleofection of purified Schwann cells with pMax-green fluorescent protein (pMax-GFP) plasmid, or with pEGFP-C-vimentin plasmid returned >45% transfection efficiency. These methods will allow the in-depth characterization of cochlear Schwann cells and an evaluation of their biochemical, functional, and genetic mechanisms that may promote neurite growth from the spiral ganglion.     

Distinctive Effects of Rat Fibroblast Growth Factor-2 Isoforms on PC12 and Schwann Cells

Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during postnatal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC 12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC 12 cells.     

Immunoliposomal Containers as Systems of Directed Transport of Minor Interfering RNA into Schwann Cells

Immunoliposomal container system for targeted delivery of minor interfering RNA into Schwann cells was developed. Monoclonal antibodies to myelin basic protein served as the vector. Analysis by sandwich ELISA of myelin basic protein showed specific suppression of the target protein gene expression in Schwann cells after incubation with PEG-conjugated immunoliposomes loaded with minor interfering RNA blocking the synthesis of myelin basic protein. Prospects of effective use of this system for targeting of minor interfering RNA for the treatment of diseases of genetic etiology are discussed. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 10, pp. 431–434, October, 2008     

体外培养Schwann细胞缺氧模型的建立

目的:建立体外培养Schwann细胞的缺氧模型。方法:取体外培养Schwann细胞,置于通入体积分数为5%CO2和95%N2的有机玻璃盒中继续缺氧培养10、15和20min,用H-E染色观察细胞形态,MTT比色试验检测细胞的活力。结果:缺氧10min组细胞形态及活力与正常组无明显差异;而缺氧15min组的细胞突起回缩,活力为缺氧前的66·3%;缺氧20min组的细胞多数死亡,活力仅为缺氧前的20·6%。结论:本方法可以成功建立有效、简便、易行的Schwann细胞缺氧模型。     

成年大鼠雪旺细胞增殖和纯化研究

目的：探索成年大鼠雪旺细胞的体外培养和纯化方法。方法：取成年大鼠背根神经节采用植块法培养，坐骨神经采用酶消化法分散培养。各分三组，第一组为阿糖胞苷处理组；第二组为免疫溶解处理后加垂体浸出液组；第三组为对照组。以活细胞计数和S-100标记相结合判断雪旺细胞增殖和纯化程度。结果：采用植块培养法至第2周末，雪旺细胞纯度在第一、二和三组分别为90％、97％和50％。分散培养至第18天，雪旺细胞纯度第一组94％，第二组大于97％，第三组80％。细胞数量第一组增加1．4倍，第二组增加5．1倍，第三组增加2．4倍。结论：采用免疫溶解处理后加垂体浸出液方法培养成年大鼠来源的雪旺细胞，可得到数量多纯度高的雪旺细胞。     

乳鼠肋间神经雪旺氏细胞培养

目的 通过12只2至4天大鼠(乳鼠)的肋间神经的雪旺氏细胞(SC)培养,获得原代雪旺氏细胞;方法 组织块反复种植法;结果 所有的肋间神经培养均能成活;结论 肋间神经是培养雪旺氏细胞的又一良好材料,在不增加成本的前提下,一只乳鼠上可获得更多的雪旺氏细胞.